Enzymes are the workhorses of our cells. Part of the key to their success is that they are able to provide supreme substrate specificity in such a chemically complex environment. Such specificity has evolved over millennia and, as such, these enzymes provide a remarkable tool for performing all manner of chemical tasks.
One group of enzymes, the methyltransferases, have evolved to catalyse the transfer of a methyl group from a small molecule cofactor, s-adenosyl-L-methionine (also called “SAM” or “AdoMet”), to a range of biomolecular targets including DNA, RNA and other proteins and enzymes. In the cell, methyltransferase-catalysed modification plays a host of important roles, such as gene regulation, and understanding of the methylation status of DNA is now a burgeoning scientific field (epigenetics).
In order to develop novel technologies based on the specific and covalent modification of biomolecular substrates by the methyltransferase enzymes, several research groups have recently developed synthetic analogues of the AdoMet cofactor. The co-factors have primarily been used with DNA methyltransferases.
Broadly, there are two classes of AdoMet: (1) aziridine derivative cofactors and (2) the so-called ‘mTAG’ cofactors carrying extended chemical chains in place of a methyl group. Unfortunately, both classes have limitations. For example, the DNA methyltransferases are unable to turn-over the aziridine derivative cofactors, meaning that stoichiometric amounts of cofactor and enzyme are required for complete substrate modification. Further, multiple enzymes have been screened for activity with these cofactors, but only a few show significant activity, perhaps due to the relatively bulky chemical structure of the aziridine analogues.
There are many reported variants of the mTAG cofactors. Examples of variants of mTAG cofactors are described in EP1874790B1. However, their synthesis is challenging and, critically, the yield of the final step of the synthesis (coupling of the transferable group to the cofactor) is low and requires the presence of several tens- to hundreds- of fold excess of the transferable moiety. This means that, for example, the coupling of a fluorophore directly to the mTAG cofactor is prohibitively expensive and scale-up to quantities of commercial relevance is challenging. Thus, there remains a need in the art for new cofactors which may be readily used with a variety of DNA methyltransferases, and for which the synthesis is straightforward and scalable.